1. Field of the Invention
The present invention provides peptides and compounds that bind to endothelial leukocyte adhesion molecule 1 (ELAM-1) and methods for blocking cell adhesion. The invention has application in the fields of biochemistry and medicinal chemistry and particularly provides ELAM-1 inhibitors for use in the treatment of human disease.
2. Description of Related Art
The accumulation of blood leukocytes at sites of inflammation depends upon the localization of these leukocytes by adhesion to the vascular lining. Certain cytokines, such as interleukin 1 (IL-1) and tumor necrosis factors alpha and beta (TNF), as well as bacterial endotoxin, have been shown to act on cultured human endothelial cells to increase leukocyte adhesion. Bevilacqua et al., December 1987, Proc. Natl. Acad. Sci. USA 84:9238-9242, incorporated herein by reference, reports the identification of an inducible endothelial cell surface protein with a molecular weight of about 115 kD, designated "endothelial-leukocyte adhesion molecule-1" (ELAM-1, also known as LECAM-2 and E-selectin) involved in the process of leukocyte adhesion to the vascular lining. Further studies have shown that this molecule is a receptor and a member of a family of inducible receptors with related structure and function on vascular cells (see Johnston et al., 24 Mar. 1989, Cell 56:1033-1044).
Hession et al., 1990, Proc. Natl. Acad. Sci. USA 87:9238-9242, incorporated herein by reference, reports the cloning and nucleotide sequence of the ELAM-1 cDNA. The sequence shows that ELAM-1 contains an N-terminal lectin domain, followed by an epidermal growth factor like domain, a number of repeating units related to those in complement binding proteins, a transmembrane domain, and a short cytoplasmic tail. The family of adhesion molecules to which ELAM-1 belongs is known as the "selectin" family and includes the proteins GMP-140 (also known as CD62, PADGEM, P-selectin, and LECAM-3) and LAM-1 (also known as LECAM-1, MEL-14, and L-selectin). See Geng et al., 22 Feb. 1990, Nature 343:757-760; Springer, 2 Aug. 1990, Nature 346:425-434; and Bevilacqua et al., 18 Oct. 1991, Cell 67:233, each of which is incorporated herein by reference). Collins et al., 5 Feb. 1991J. Biol. Chem. 266(4):2466-2473, reports the nucleotide sequence of the ELAM-1 gene, which contains 14 exons spanning about 13 kb of DNA on chromosome 1.
The lectin motif of ELAM-1 is believed to be involved in the binding of carbohydrate ligands, and ELAM-1 has been shown to bind to sialyl-Lewis X (SLe.sup.x), a terminal structure found on cell surface glycoprotein and glycolipid carbohydrate groups of neutrophils, during leukocyte adhesion (see Phillips et al., 23 Nov. 1990, Science 250:1130-1135; Parekh, 1991, Oxford GlycoSystems Ltd., Tech. Bull. 11; Tiemeyer et al., Feburary 1991, Proc. Natl. Acad. Sci. USA 88:1138-1142; and Tyrrell et al., November 1991, Proc. Natl. Acad. Sci. USA 88: 10372-10376, each of which is incorporated herein by reference). Other selectins, such as the leukocyte receptor CD62, also recognize the SLe.sup.x ligand (see Polley et al., July 1991, Proc. Natl. Acad. Sci. USA 88: 6224-6228, incorporated herein by reference). Other workers have reported the isolation of a cDNA that directs the expression of an ELAM-1 ligand. The cDNA encodes a 46 kD protein that has alpha(1,3)fucosyltransferase activity, suggesting that a fucosylated carbohydrate structure is an essential component of the ELAM-1 ligand (see Goelz et al., 21 Dec. 1990, Cell 63:1349-1356).
The ligands for ELAM-1 are apparently present on memory T cells, because Shimizu et al., 28 Feb. 1991, Nature 349:799-802, reports that ELAM-1 may be of primary importance in the initial attachment of memory T cells to inflamed endothelium in vivo and to the preferential migration of memory T cells into tissue and inflammatory sites. Picker et al., 28 Feb. 1991, Nature 349:796-799, reports that ELAM-1 may function as a skin vascular addressin, a tissue selective endothelial cell adhesion molecule for skin homing memory T lymphocytes. Although ELAM-1 undoubtedly serves important biological functions, the inappropriate expression of ELAM-1 can be very detrimental to the host. For instance, inappropriate production of or response to IL-1, a cytokine that also stimulates transient expression of ELAM-1, plays a role in many chronic inflammatory diseases, such as rheumatoid arthritis (RA), osteo arthritis (OA), psoriasis, inflammatory bowel disease, encephalitis, glomerulonephritis, and respiratory distress syndrome. See Bender and Lee, 1989, Ann. Rep. Med. Chem. 25:185-193; and U.S. Pat. No. 5,075,222, particularly columns 1 to 3, each of which is incorporated herein by reference.
By inhibiting the binding of leukocytes to ELAM-1, ELAM-1 inhibitors can be used to ameliorate the effects of inappropriate production or response to ELAM-1 or the cytokines that stimulate ELAM-1 production, such as the cytokines TNF or IL-1. Furthermore, ELAM-1 has been reported to promote tumor cell adhesion and so may be involved in cancer metastases (see Rice and Bevilacqua, 8 Dec. 1989, Science 246:1303-1306, incorporated herein by reference), so ELAM-1 antagonists may be beneficial in the treatment of cancer, acting to prevent metastases.
Scientists have demonstrated increased levels of ELAM-1 in (1) dermal inflammation, including Il-1-induced skin inflammation; atopic dermatitis, psoriasis, and allergic contact dermatitis; delayed-type hypersensitivity reaction induced by DNCB on human skin; and endotoxin-induced acute cutaneous inflammation in baboons; (2) intestinal inflammation, including inflamed colonic mucosa of patients with ulcerative or Crohn's colitis; inflammatory bowel disease; and intestinal graft-versus-host disease; (3) other sites of inflammation, including synovial tissues from patients with RA and OA; rat endotoxin-induced uveitis, and inflamed endothelium of asthma model (primate antigen inhalation); and (4) septic shock induced in baboons with live E. coli. Because ELAM-1 inhibitors can be used to inhibit the deleterious effects of inflammation, various companies have been reported to be testing such compounds for various applications. These compounds include: (1) monoclonal antibodies against ELAM-1 under testing by Otsuka; Cytel; and Biogen for treatment of acute and chronic inflammatory diseases; (2) carbohydrate inhibitors of ELAM-1 under testing by Glycomed; Cytel; and Oxford GlycoSystems/SmithKline Beecham, for inflammatory diseases and cancer; and (3) other compounds, including antisense oligonucleotides, under testing by Isis for treatment of inflammation and cancer.
The availability of cloned genes for ELAM-1, including a soluble ELAM-1 derivative, facilitates the search for agonists and antagonists of this important receptor. The availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems, even though the natural ligands for ELAM-1 appear to be carbohydrates. These systems include the "peptides on plasmids" system described in U.S. patent application Ser. No. 963,321 filed Oct. 15, 1992, which is a continuation-in-part of Ser. No. 778,233, filed Oct. 16, 1991, the "peptides on phage" system described in U.S. patent application Ser. No. 718,577, filed Jun. 20, 1991, and in Cwirla et al., August 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382, the "encoded synthetic library" (ESL) system described in U.S. patent application Ser. No. 946,239, filed Sep. 16, 1992, which is a continuation-in-part of Ser. No. 762,522, filed Sep. 18, 1991, and the "very large scale immobilized polymer synthesis" system described in U.S. Pat. No. 5,143,854; PCT patent publication No. 90/15070, published Dec. 13, 1990; U.S. patent application Ser. No. 624,120, filed Dec. 6, 1990; Fodor et al., 15 Feb. 1991, Science 251:767-773; Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271-180; and U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991; each of the foregoing patent applications and publications is incorporated herein by reference.
There remains a need for non-carbohydrate compounds that bind to or otherwise interact with ELAM-1 with high affinity, both for studies of the important biological activities mediated by this receptor and for treatment of disease. In similar fashion, there remains a need for compounds that bind to ELAM-1 and block the binding of leukocytes to ELAM-1. The present invention provides such compounds.